Many forms of serious human disease result from an autoimmune attack on the body. Many forms of autoimmune disease, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and Crohn's disease, are particularly difficult to treat. An activated CD4+ T cell that releases interleukin-17 (IL-17), a powerful pro-inflammatory cytokine (Aggarwal, Ghilardi et al. 2003), and is referred to as a TH-17 cell (Bettelli, Carrier et al. 2006; Mangan, Harrington et al. 2006; Veldhoen, Hocking et al. 2006), may be a significant pathogenic factor autoimmune disease, based on observations that patients have high levels of IL-17 expression in target tissues. For example, the level of IL-17 mRNA was found to be elevated in brain autopsy tissue from patients with MS compared with controls, and the number of IL-17 positive mononuclear cells was also increased in the cerebrospinal fluid of affected patients (Matusevicius, Kivisakk et al. 1999; Lock, Hermans et al. 2002; Vaknin-Dembinsky, Balashov et al. 2006). Rheumatoid arthritis (RA) also appears to involve a T cell-mediated autoimmune reaction. The level of IL-17 mRNA in synovial fluid of rheumatoid arthritis patients is predictive of disease progression (Kirkham, Lassere et al. 2006). Furthermore, TH-17 cells have been isolated from intestinal biopsies of patients with Crohn's Disease (Annunziato, Cosmi et al. 2007), and their frequencies are elevated in comparison to T cells isolated from normal intestine or peripheral blood. In disease tissue of psoriasis patients, levels of IL-22, another proinflammatory cytokine produced by TH-17 cells, are elevated (Zheng, Danilenko et al. 2007).
TH-17 cells, also referred to as IL-17+CD4+ T or TIL-17 cells, have only been recently characterized and are distinct from the TH1 and TH2 lineages of CD4+ effector T cells. The relationship of TH-17 cells to the TH1 and TH2 lineages is diagrammed in FIG. 1. TH1 and TH2-derived cytokines (such as IFN-γ and IL-4) block TH-17 formation (FIG. 1) and targeted deletion of some transcription factors considered to be central for maintenance of the TH1 and TH2 phenotype have no major effect on TH-17 differentiation (Harrington, Hatton et al. 2005; Park, Li et al. 2005). IL-23 expression in mouse is required for induction of T cell-derived IL-17, and in culture IL-23 seems to act as a survival factor for pre-existing TH-17 cells but does not stimulate TH-17 formation from naïve CD4+ T cells. Instead, the combination of TGFβ and IL-6 is required to stimulate the formation of TH-17 cells from naive mouse CD4+ T cells, and there is genetic evidence in mouse for the involvement of IL-6 and TGFβ in TH-17 formation in vivo (Bettelli, Carrier et al. 2006; Mangan, Harrington et al. 2006; Veldhoen, Hocking et al. 2006). The cytokine requirements for TH-17 differentiation in human are somewhat different that in mouse but the TH-17 phenotype is similar, including expression of IL-17, IL-22, and RORγ (Annunziato, Cosmi et al. 2007; Kebir, Kreymborg et al. 2007).
The older view that autoreactive CD4+ TH1 cells are a prime cause for the development or progression of MS and other forms of autoimmune disease has been challenged (Steinman 2007) by recent results from rodent models showing that mice with a targeted deletion in the p35 subunit of IL-12, which is required for the formation of TH1 cells, are still highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) after immunization with myelin antigens (Becher, Durell et al. 2002; Gran, Zhang et al. 2002). In contrast, the knockout of either p19, the unique subunit of the cytokine IL-23, or p40, a common subunit of the two cytokines IL-12 and IL-23, create mice that are resistant to the induction of EAE (Cua, Sherlock et al. 2003; Langrish, Chen et al. 2005).
In addition to EAE, development of disease in a mouse model of rheumatoid arthritis, collagen-induced arthritis (Courtenay, Dallman et al. 1980), is also dependent on IL-23 function and is correlated with an increased level of TH-17 cell activity, such as release of IL-17 in affected tissues (Murphy, Langrish et al. 2003; Sato, Suematsu et al. 2006). Inhibition of IL-17 may be a viable alternative to inhibition of TNFα in treatment of RA (Lubberts, Koenders et al. 2005). Further, IL-23 expression is required for induction of IBD in mouse, and TH-17 cells appear to be an important downstream mediator of IL-23 effect in this model (Yen, Cheung et al. 2006). Disease progression in EAE models is partially suppressed in IL-17−/− mice or after treatment with anti-IL-17 antibodies (Iwakura and Ishigame 2006). IL-17−/− mice also show reduced incidence and severity in a collagen-induced arthritis (CIA) model for rheumatoid arthritis and IL-17 antibodies have been shown to attenuate development of intestinal inflammation in rodent models of IBD (Nakae, Nambu et al. 2003; Yen, Cheung et al. 2006). Further, purified autoreactive TH-17 cells strongly induce encephalomyelitis when transferred to naive mice, leading to more dramatic disease manifestation compared to disease induction by TH1CD4+ T cells (Langrish, Chen et al. 2005; Komiyama, Nakae et al. 2006). The IL-27 receptor is prominently expressed in TH-17 cells and the action of IL-27 as an inhibitor of murine EAE is correlated to reduced TH-17 cell number in the animal (Batten, Li et al. 2006; Stumhofer, Laurence et al. 2006). Therefore, the activity of murine TH-17 cells is well correlated to disease.
Inhibition of the human equivalent of the mouse TH-17 cell is a highly desirable target for new therapeutic agents to treat autoimmune disease. Recent findings implicate TH-17 cells in the pathogenesis of Crohn's disease and more generally inflammatory bowel disease (IBD). Antibody to the common subunit of IL-23 and IL-12, p40, is safe and may be clinically effective in treatment of Crohn's disease (Mannon, Fuss et al. 2004). A recent Phase 2 clinical trial also shows that anti-p40 is highly effective in the treatment of psoriasis (Krueger, Langley et al. 2007). The identification of TH-17 cells has not only provided insight into autoimmune pathogenesis, it has also revealed a major pathway of adaptive immunity for extracellular microbes (Mangan, Harrington et al. 2006; Annunziato, Cosmi et al. 2007). Stimulation of TH-17 differentiation, function and cytokine release therefore has the potential to enhance protective immunity by increasing T cell reactivity to pathogenic organisms and other targets, such as cancer-associated antigens.
Structure and Function of RORγ
RORγ (NR1F3), a ligand-regulated nuclear transcription factor from the steroid/retinoid/thyroid family (Jetten, Kurebayashi et al. 2001), has been shown to be essential for CD4+ TH-17 development and/or function. RORγ participates in and is required for the development of TH-17 cells. TH-17 cells are absent from genetically-engineered mice that fail to express a specific splicing isoform of RORγ, RORγt (Ivanov, McKenzie et al. 2006; Littman and Eberl 2006). Furthermore, an RORγt-GFP transgene that expresses GFP from the RORγt promoter is expressed in CD4+IL-17+ T cells from the lamina propria of the gut and other tissues. In cell culture, transfection of RORγt into naïve murine CD4+ T cells induces differentiation of these cells into IL-17 expressing T cells even in the absence of the inducing cytokines IL-6 and TGFβ. The data suggest that the transcriptional activity of RORγt is of major importance to TH-17 cell differentiation and function (Ivanov, McKenzie et al. 2006). RORγt expression is induced in the presence of TGFβ and IL-6 or by TGFβ and IL-21, an autocrine cytokine released from developing TH-17 cells in response to IL-6 (Ivanov, McKenzie et al. 2006; Korn, Bettelli et al. 2007; Nurieva, Yang et al. 2007; Zhou, Ivanov et al. 2007).
The expression of RORγ follows a similar pattern in human as in mouse T cells: RORγ is more highly expressed in IL-17 or IL-17/IFNγ expressing CD4+ T cells (Th-17) than in CD4+ T cells that express IFNγ alone (Th-1) (Acosta-Rodriguez, Napolitani et al. 2007; Acosta-Rodriguez, Rivino et al. 2007; Annunziato, Cosmi et al. 2007; Wilson, Boniface et al. 2007). Finally, it has been reported that other murine T cell types express RORγt, including γδTCR+ cells, CD8+ T cells, and iNKT cells (Ivanov, McKenzie et al. 2006; Ivanov and Littman 2007). Since these cells are also express IL-17, it is possible that RORγt is required for the differentiation of the IL-17+ subpopulations of several other types of T cells.
Finally, in the absence of RORγt, mice are much less susceptible to the induction of EAE (Ivanov, McKenzie et al. 2006). These studies were carried out in immunodeficient mice. RORγt−/− mice lack lymph notes, and although they are resistant to the development of EAE, a further study was carried out by adoptive transfer of RORγt wild type or RORγt−/− bone marrow to immunodeficient mice. While transfer of normal bone marrow rendered the mice sensitive to the induction of EAE by peptide immunization, the RORγt−/− transfectants were substantially resistant (Ivanov, McKenzie et al. 2006). These data suggest that RORγt mediated regulation of T cell differentiation is involved in the development of autoimmune disease.
RORγStructure. RORγ, like other members of the nuclear receptor family, has a bipartite structure with two major functional domains, a DNA binding domain (DBD) and a ligand binding domain (LBD, see FIG. 2) (Medvedev, Yan et al. 1996). Ligand-regulated transcription of the nuclear receptors is mediated through the LBD, which has been crystallized for many receptors (Li, Lambert et al. 2003), including RORα and RORβ, the receptors most closely related to RORγ. The RORγ LBD is predicted to have a binding pocket similar to RORα and RORβ (Stehlin, Wurtz et al. 2001; Kallen, Schlaeppi et al. 2004). The retinoic acid receptors (RARα, RARβ, and RARγ) are more distantly related and appear not to have a functional overlap with the RORs (Jetten, Kurebayashi et al. 2001).
The nuclear receptor LBD recruits transcriptional coregulators (FIG. 2) in response to small molecule compounds (Li, Lambert et al. 2003; Savkur and Burris 2004). These coregulatory proteins act as sensors for the conformational state of the ligand-bound complex and in turn regulate the recruitment of transcriptional factors to chromatin adjacent to the receptor. Short peptide domains of the coregulatory proteins required for interaction with the nuclear receptor contain the conserved sequence LXXLL (SEQ ID NO: 1), and synthetic peptide recruitment assays based on this motif are widely used to monitor the binding of agonists and antagonists to the nuclear receptor LBD (Lee, Elwood et al. 2002; Savkur and Burris 2004), including for an assay described herein for RORγ.
Functional Studies of RORγ.
Recognition of specific DNA motifs by the nuclear receptor DBD determines specificity for gene transcription; however, little is known about specific gene targets for RORγ, and the major findings on RORγ function have been derived from studies of gene-targeted mice (Kurebayashi, Ueda et al. 2000; Sun, Unutmaz et al. 2000; Eberl and Littman 2004; Eberl, Marmon et al. 2004). RORγ has two splicing isoforms, RORγ and RORγt (He, Deftos et al. 1998). RORγt differs from RORγ by a truncation of 21 amino acids at the N-terminal and is the isoform specifically expressed in thymus, lymph node precursors, and TH-17 cells (Eberl and Littman 2004; Eberl, Marmon et al. 2004; Ivanov, McKenzie et al. 2006; Littman and Eberl 2006), the major tissues affected in knockout studies of RORγ. The significance of the N-terminal deletion of RORγ is not known, but it is unlikely to affect ligand specificity or LBD function, which is encoded at the receptor's C-terminal and is identical in the two splicing isoforms.
In addition to its requirement for TH-17 differentiation, RORγ has other discrete functions in the immune system. In its absence, the survival of the major subtype of developing T lymphocytes, the CD4+CD8+ double positive (DP) thymocytes, is reduced (Kurebayashi, Ueda et al. 2000; Sun, Unutmaz et al. 2000), and the embryonic formation of lymph nodes and Peyer's patches is blocked (Eberl, Marmon et al. 2004). RORγt is an early marker for the embryonic formation of lymphoid tissue inducer or LTi cells. LTi cells are involved in lymph node and Peyer's patch formation during embryogenesis (Eberl, Marmon et al. 2004). After birth, an LTi-like cell participates in formation of intermediate lymphoid follicles (ILFs) of the gut. These lymphoid structures appear to participate in the gut immune response (Eberl and Littman 2004). RORγ is also expressed in liver, muscle, and fat (Jetten, Kurebayashi et al. 2001; Fu, Sun et al. 2005). A recent study of RORγ−/− mice also suggests that the receptor has some regulatory effects on Phase 1 and Phase 2 detoxification enzymes (Kang, Angers et al. 2007).
Clinical Significance.
Three major points of action of RORγ in the immune system have been identified in gene knockout studies; T cells, including the TH-17 cell, lymph node formation, and survival of DP thymocytes. Of these, regulation of IL-17+ T cell function, including TH-17 differentiation, appears to be most relevant to human therapeutics. Not only is the receptor absolutely required for TH-17 differentiation, but the supply of pathogenic TH-17 cells must be constantly replenished since they are destroyed in target tissue (Gold, Linington et al. 2006). Inhibition of new TH-17 formation will therefore have important benefits. RORγ is expressed in human memory TH-17 cells (Acosta-Rodriguez, Rivino et al. 2007; Annunziato, Cosmi et al. 2007), and data presented herein shows that RORγ antagonists block IL-17 expression in human peripheral blood mononuclear cells (PBMCs). The primary source of IL-17 in PBMCs has been reported to be memory T cells (Shin, Benbernou et al. 1999). Finally, RORγ enhances, but is not absolutely required for, the survival of the major developing T cell type of the thymus, the DP thymocyte. In the knockout animal, there is no evidence of immunodeficiency although splenic and peripheral frequencies of some immune cell types are changed. However, there is a much higher rate of apoptosis among thymocytes and thymocyte number is reduced (Kurebayashi, Ueda et al. 2000; Zhang, Guo et al. 2003). The thymus of an adult has already atrophied to a considerable degree and can be reversibly suppressed by many forms of pharmacological treatment, including exposure to steroids (Haynes, Markert et al. 2000). These data suggest that secondary effects of an RORγ antagonist on thymic function in human may not be clinically significant.
Prior to 1990, no drugs to treat MS were available. In the last 15 years, several treatment options have emerged, primarily various forms of INFβ (Avonex, Rebif, Betaseron), Glatiramer acetate (Copaxone) and the chemotherapeutic drug mitoxantrone (Novantrone) (Rolak 2003). INFβ and Glatiramer acetate both appear to inhibit T cell activation and both drugs reduce the number of attacks in relapsing-remitting MS but have little effect in the progressive phase of the disease (Dhib-Jalbut 2002; Rolak 2003). The long-term benefits of these drugs are unclear. Mitroxantrone appears to retard progression and delay disability in secondary progressive MS. However, toxicity of this drug is very limiting and Mitroxantrone is considered a short-term treatment option. More recently, a humanized antibody to α4β1 integrin (NATALIZUMAB, Tysabri) has been approved for the treatment of MS and has been shown to slow disease progression and reduce relapse rate in several clinical trials using different outcome measures (Steinman 2005). Compared to existing treatments, the efficacy of Tysabri is quite dramatic. However, several cases of progressive multifocal leukoencephalopathy (PML), a lethal resurgence of a latent viral infection linked to the immunosuppressive action of the drug, led to withdrawal (Rudick, Stuart et al.). Tysabri has now been reintroduced into the market with much stricter patent monitoring. Tysabri likely blocks both TH-17 and TH1 cells. A novel small molecule drug that is specific for TH-17 cells and does not compromise the antiviral activity of TH1 cells could be safer and more effective for several reasons: (1) a small molecule drug, administered daily, can be rapidly withdrawn if significant side effects occur; (2) a small molecule drug is more readily manufactured and more easily administered than a biologic, such as Tysabri; and (3) TH1 cells suppress TH-17 differentiation and thus specific inhibition of TH-17 cells may be more effective.
A number of other small molecule drugs are marketed for autoimmune diseases such as rheumatoid arthritis, IBD, and psoriasis. Many of these, such as methotrexate or azathioprine, carry significant toxicity because of anti-metabolite or anti-mitotic effects. Dosing is usually limited. A small molecule drug that has a more specific mechanism of action, such as inhibition of TH-17 cells or other IL-17 expressing T cells, are likely to be safer and, hence, more efficacious as dosages may be elevated to have a substantial inhibitory effect on target cells.
Pharmacologically useful ligands to members of the ROR family of orphan nuclear receptors have not been identified in the published literature. Cholesterol and cholesterol sulfate occupy the ligand binding pocket within the receptor LBD of RORα as determined by x-ray crystallography (Kallen, Schlaeppi et al. 2002; Kallen, Schlaeppi et al. 2004), but, due to the fact that these molecules are plentiful in normal cells, it has not been possible to use these molecules in order to characterize RORα as a pharmacological target (Moraitis and Giguere 2003). A series of RORα ligands was published in 1996 (Missbach, Jagher et al. 1996; Wiesenberg, Chiesi et al. 1998), but these findings have not been independently replicated or evaluated by functional criteria described below. One of the proposed ligands for RORβ, melatonin, has been challenged in the literature (Becker-Andre, Wiesenberg et al. 1994; Greiner, Kirfel et al. 1996; Becker-Andre, Wiesenberg et al. 1997). More recently, it was proposed that all-trans retinoic acid and the synthetic retinoid ALRT 1550 are functional ligands for RORβ and that these two ligands also regulate RORγ in a similar manner, in both cases inhibiting the transcriptional activity of the receptors. All-trans retinoic acid and ALRT 1550 were referred to as “functional” ligands because they were presumed to both bind and regulate transcription through RORβ. The ligands are unlikely to be useful pharmacologically because they are potent activators of the retinoid receptors, RARα, RARβ, RARγ (Thacher, Vasudevan et al. 2000). Therefore all of these ligands fail the test of functional usefulness either on the criterion that their effects have not been reproducible, or because they are ubiquitous, or because they lack specificity. This application describes assay for RORγ, and RORγ ligands that have pharmacologically useful potency (in the range of 50 nm to 1 that have good selectivity, as demonstrated by assays for other members of the nuclear receptor family. These ligands, both agonists and antagonists, have drug-like properties as indicated by rational structure activity relationships among analogues as well as other drug-like properties such as bioavailability and activity in cellular and animal models. Specific RORγ antagonists are predicted to be highly useful in treatment of autoimmune disease by blocking TH-17 function. Agonists of RORγ are predicted to enhance immunity and to have application in stimulation of vaccination and in adjuvant cancer therapy.